Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion.Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion,but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear.Therefore,in the current study,we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons.Hippocampal neurons were treated with Mg^2+-free extracellular solution containing glutamate(300μM)for 3 hours as a model of glutamate-mediated excitotoxicity(glutamate group).In the normal group,hippocampal neurons were incubated in Mg^2+-free extracellular solution.In the Achyranthes bidentata polypeptide group,hippocampal neurons were incubated in Mg^2+-free extracellular solution containing glutamate(300μM)and Achyranthes bidentata polypeptide at different concentrations.At 24 hours after exposure to the agents,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear m'orphology,respectively.Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay,respectively.At various time points after glutamate treatment,reactive oxygen species in cells were detected by H2 DCF-DA,and mitochondrial membrane potential was detected by rhodamine 123 staining.To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors,electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons.Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate.In addition,Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current.Furthermore,the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment.These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway.All animal studies were approved by the Animal Care and Use Committee,Nantong University,China(approval No.20120216-001)on February 16,2012.

Facile Preparation of Polypeptides:Moisture Insensitive and Superfast Ring Opening Polymerization of N-Carboxyanhydrides

(a)NCA polymerization initiated by LiHMDS or other initiators in THF,initiator i)n-hexylamine,ii)HMDS,iii)bipyNi(COD);(b)LiHMDS-initiated open vessel polymerization of BLGNCA at 26 mg and 2 g scale;(c)GPC traces of poly-BLG at variable DP;(d)Reaction rates of LiHMDS and hexylamine initiated BLGNCA polymerization in THF with NCA:initiator ratio of 100∶1 and initial NCA concentration at 0.2 mol/L;(e)CD spectra of poly-BLG at variable DP prepared from LiHMDS-initiated NCA polymerization.

Force-Regulated Adhesion and Activation Study of Integrin Targeting Polypeptides

Integrins are heterodimeric cell surface receptors that bind to ligands on another cell,e.g.intercellular adhesion molecule 1(ICAM-1),or the extracellular matrix.Integrins play an important role in immune system,and they participate in inflammation,thrombosis,and proliferation,migration and apoptosis of tumor cells.They mediate adhesion and transduce signals across the membrane usually under the influence of forces.A recent study has shown that integrins bind and activate transforming growth factorβisoform(TGF-β)which is involved in tumor suppression and growth,and blocking the binding of TGF-βto integrin can inhibit tumor growth.RGD(arginine-glycine-aspartate)small peptide,which competitively inhibits ligand binding to integrins,has been approved as an injectable drug.However,when the RGD is used to block cancer-related extracellular signaling pathways,it will also cause activation of integrins for a period,and stimulate the transduction of intracellular signals constantly.Therefore,it is necessary to explore for new drugs that can selectively control conformational state of integrins without activating or blocking all of them.In this study,we selected two small peptides,KQAGDV and RTDLDSLRT,that combined with integrins and do not contain an RGD sequence.The non-RGD polypeptide RTDLDSLRT has been reported to have a binding site with integrins and the binding affinity is on nanomolar scale.For the motif of the fibrinogen y chain C-terminal KQAGDV,it can adhere to the head of the integrins.The micropipette aspiration technique and electron microscopy techniques were used to study the adhesion and activation of integrins by peptides,respectively.Micropipette aspiration technique was used to investigate the adhesion frequency of peptide and integrin on Jurkat cell.The pressure system was used to supply a controllable negative pression to the microtube,and two micropipettes were used to absorb red blood cells and Jurkat cells,respectively.The red blood cells were coated with small peptides and can serve as a force sensor after being sucked when two cells were connected.The binding kinetics of integrin and peptides interactions was determined by fitting the curves constructed using adhesion probability between two cells as a function of time.The curves were fitted using a small system probabilistic kinetic model to estimate a pair of kinetic parameters,including the zero force reverse rate kr0,and the cellular binding affinity Acmrm1Ka0.The adhesion frequency yielded P(t)=75%and 57%for RGD and KQAG DV peptides,respectively.We obtained Acmrm1Ka0=1.40 and kr0=0.32 s-1,for RGD,and Acmrm1Ka0=0.85 and kr0=0.54 s-1 for KQAGDV.The RGD peptide has a higher adhesion frequency and lower dissociation rate than the KQAGDV peptide.Electron microscopy techniques was used to observe the activation of integrins by peptides.Jurkat cell expressing integrins was bound to a magnetic bead and bottom plate which were coated with different integrin-binding peptides.Then,we manipulated the beads in a controlled direction by changing the magnetic field nearby,and the forces were applied to the cell.The target cells were fixed and then observed by scanning electron microscope or transmission electron microscope.Jurkat cells contain abundant flexible microvilli of which there are many parallel bundles of actin filaments inside.By electron microscopy analysis,the cell connected with magnetic bead coated with RGD were found to be protruded and the size of microvilli increased up to#-fold of the length of the KQAGDV sample.The microvilli exhibited a curved agglomerate structure under a force-free condition.Moreover,a higher proportion of cells were activated in the presence of RGD than KQAGDV.In conclusion,the binding affinity of KQAGDV to integrin is weaker than RGD,and KQAGDV can bind with integrins effectively with a lower activated proportion.Our results indicate the peptides may selectively bind to integrins without activating them.

Protective effects of Achyranthes bidentata polypeptides on retinal ganglion cells post-optic nerve crush in rats

Achyranthes bidentata polypeptides（ABPP） have been reported to inhibit apoptosis of retinal ganglion cells（RGCs）.The present study investigated the protective effects of ABPP on RGCs in a rat model of optic nerve injury.With prolonged injury time,RGC densities were gradually decreased.ABPP（5 μg） significantly increased RGC densities and upregulated growth associated protein 43 expression in rats with optic nerve injury.Results demonstrate that ABPP can protect RGCs and promote axonal growth after optic nerve crush.

Cationic polypeptides in a concept of oppositely charged polypeptides as prevention of postsurgical intraabdominal adhesions

Background: Two differently charged polypeptides, α-poly-L-lysine and poly-L-glutamate, have previously been shown to effectively reduce postoperative intraabdominal adhesions. Though α-poly-L-lysine showed toxicity in doses too close to the lowest therapeutic dose, the aim in the present study was to investigate the possible antiadhesive effect of another four cationic polypeptides. Materials/Methods: 125 mice were studied with a standardized and reproducible adhesion model and given epsilon poly-L-lysine, lactoferrin, lysozyme and polyarginine respectively in a combination with poly-L-glutamate. Epsilon poly-L-lysine was also tested in different concentrations and as single treatment. Results: All four cationic polypeptides above showed a significantly better anti-adhesive effect than the controls receiving saline (p<0.05). Epsilon poly-L-lysine had the best antiadhesive effect of the new substances tested in the experiment. Single treatment with the epsilon poly-L-lysine showed toxic side effects. Discussion: We have shown that epsilon poly-L-lysine, polyarginine, lysozyme and lactoferrin, in descending order, all can reduce postoperative intraabdominal adhesions in mice when combined with poly-L-glutamate. There were side effects of epsilon poly-L-lysine resembling those of α-poly-L-lysine, although less toxic. The antiadhesive effect of epsilon poly-L-lysine did not reach the level of α-poly-L-lysine. Further studies will concentrate on additional investigation, trying to modify the α-poly-L-lysine to lower its toxicity. The less toxic epsilon poly-L-lysine also needs further attention in our research of antiadhesive bioactive polypeptides.

SURFACE MODIFICATION OF POLYPROPYLENE MICROPOROUS MEMBRANE BY TETHERING POLYPEPTIDES

Two kinds of polypeptides were tethered onto the surface of polypropylene microporous membrane （PPMM） through a ring opening polymerization of L-glutamate N-carboxyanhydride initiated by amino groups which were introduced by ammonia plasma and y-aminopropyl triethanoxysilane treatments. X-ray photoelectron spectroscopy （XPS）, attenuated total reflectance Fourier transform infrared spectroscopy （FT-IR/ATR）, scanning electron microscopy （SEM）, together with water contact angle measurements were used to characterize the modified membranes. XPS analyses and FT-IR/ATR spectra demonstrated that polypeptides are actually grafted onto the membrane surface. The wettability of the membrane surface increases at first and then decreases with the increase in grafting degrees of polypeptide. Platelet adhesion and murine macrophage attachment experiments reveal an enhanced hemocompatibility for the polypeptide modified PPMMs. All these results give evidence that polypeptide grafting can simultaneously improve the hemocompatibility as well as reserve the hydrophobicity for the membrane, which will provide a potential approach to improve the performance of polypropylene hollow fiber microporous membrane used in artificial oxygenator.

Synthesis and Cleavage Activity of Artifical Minic Polypeptides

Two artificial minic polypeptides which are synthetic analogues of natural products with DNA affinity were synthesized, and theirs cleavage activity with DNA were examined. The structures of these compounds was confirmed by ^1H NMR, MS and IR.

Construction of Expressing Plasmids of Recombinant FN Polypeptides with Bifunctional－domain and the Characterization of the P

Two expressing plasmids have been constructed and used to express two bifunctional-domain recombinant polypeptides of human fibronectin (FN) in E. coli. One was CH50 (Pro1239-Ser1515 of FN linked with Ala1690-Thr1960 of FN through Met) and the other was CH56 (Pro1239-Thr1960 of FN). Both of two polypeptides were capable of binding heparin and were purified by heparin-a-garose affinity chromatography. The purified products were capable of binding cells. The production of CH50 and CH56 polypeptides provided a fundamental basis for further study of the anti-metastatic function of recombinant fibronectin polypeptides.

Structure Characterization of Silk Fibroin Crystalline Domain Polypeptides Expressed in Escherichia coli

The molecular conformations of four silk fibroin crystalline analogues [GAGAG-X] 16(G,Gly;A,Ala;X=Ala,Ser,Tyr or Val,designated eGA,eGS,eGY or eGV),carried out using molecular design and expressed by Escherichia coil(E.coli),were evaluated by Raman spectra analysis.The abilities of forming β-sheet structure were determined by thioflavin T(ThT) fluorescence spectra analysis.In terms of molecular conformation,except eGY that could not form significant typical molecular conformation,eGS and eGV were mainly composed of β-sheets while eGA tended to form β-turn.β-turn was also present in eGY and absent in eGS and eGV.In terms of β-sheet structure,eGS had the highest β-sheet content,followed by eGV,and eGA had the lowest content,furthermore,β-sheet structures were more stable in eGS and eGV than those in eGA and eGY.

Therapeutic effects of velvet antler polypeptides on hepatic fibrosis in rats

OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides(VVAPs)in CCl4-induced experimental hepatic fibrosis in rats.METHODS Anti-hepatic fibrosis properties of VAPs were tested by Subcutaneous injection(SC)into male Wistar rats of CCl4- induced experimental hepatic fibrosis.After SC injections for 45 consecutive days at doses of 5mg·kg-1(low dose,VAPsL),10mg·kg-1(mid-dose,VAPsM)and 20mg·kg-1(high-dose,VAPsH),the rats were sacrificed and the various indicators were evaluated and tested.Observed hepatic cells degeneration and necrosis,inflammatory infiltration and levels of serum enzymes to assess treatment of VAPs;The expression levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),MDA,and hydroxyproline(HYP)in liver tissue were analyzed;RT-PCR analysis was carried out to detect the expression levels of matrix metalloproteinases2(MMP-2)and tissue inhibitor of metalloproteinases 1(TIMP-1)in liver tissue.RESULTS VAPs has obvious anti-hepatic fibrosis effects.Hepatocyte swelling,fatty degeneration was significantly reduced,reducing infiltration of inflammatory cells.Release of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)decreased significantly,reduction of hyaluronic acid(HA)and laminin(LN)obviously,at the same time,the content of total protein and albumin increased significantly in serum.Activity of SOD and GSH-Px was significantly raised and the content of MDA and HYP was reduced significantly in liver tissue.Expression levels of MMP-2and TIMP-1 mRNA in liver were decreased significantly.These improvements were more significant in high-dos and mid-dose groups(P<0.05 or P<0.01 vs model group).CONCLUSION These findings suggest VAPs can significant treat the hepatic fibrosis,which may be due to protect liver cells and improve liver functions by hydroxyl radical scavenging activity and great effect of antioxidation,and decrease the gene expression of MMP-2,improving exist-environment of liver cells and decreasing the gene expression of TIMP-1,prompting degradation of extracellular matrix.

Alteration of Crystallin Polypeptides in Rat Lenses during the Development of Galactose-induced Cataract

Some striking differences in relative polypeptide abundanceof crystallins were observed in normal and galactose-induced cataractouslenses of rat by means of SDS-PAGE.In the cataractous lenses aprominent band appeared at about 25 kDa and the αA chain increasedmarkedly,whereas the relative amount of the 31 kDa band decreasedsubstantially.These alterations are similar to the changes observed duringthe incubation of young mouse lenses in glucose-free medium.Eye Science1993;9:143-145.

Polypeptides inhibit HIV-1 replication by interfering viral Vpu-mediated tetherin degradation

Background:HIV-1 Vpu acts by counteracting the tethering function of tetherin and resulting in the release of HIV-1 virion.Disrupting Vpu-tetherin interactions may provide a promising new target for antiretroviral therapy.Methods:Polypeptides that covered the amino acid sequence on the interface of Vpu-tetherin complex were designed.Phenotypic susceptibilities and cellular toxicities to the polypeptides were measured.The mechanisms of the anti-HIV-1 polypeptides were determined by the Western blot analysis and laser confocal scanning.Seven 20-mer polypeptides from wild-type Vpu amino acid sequence were designed.Results:We report the design and identification of 3 novel anti-HIV-1 polypeptides that derived from Vpu se-quence which can efficiently inhibit HIV-1 infection.A pilot mechanism study showed that the active polypeptide could counteract Vpu-mediated tetherin downregulation.Laser confocal image scanning study showed that the polypeptides bound on the cell surface with a receptor specific binding manner,which may target tetherin that expressed on cell surface.Conclusion:Our work provided first evidence that counteracting Vpu-mediated tetherin downregulation could be a target for novel anti-HIV-1 drug design.Future works to provide direct evidence of inhibitors interact with teth-erin at atomic resolution and the development of small molecules inhibitors targeting Vpu-tetherin interactions may open a new avenue for novel antiretroviral therapy.

Targeted screening of an anti-inflammatory polypeptide from Rhopilema esculentum Kishinouye cnidoblasts and elucidation of its mechanism in alleviating ulcerative colitis based on an analysis of the gut microbiota and metabolites

Ulcerative colitis(UC)is a recurrent inflammatory bowel disease that imposes a severe burden on families and society.In recent years,exploiting the potential of marine bioactive peptides for the treatment of diseases has become a topic of intense research interest.This study revealed the mechanism underlying the protective effect of the dominant polypeptide PKKVV(Pro-Lys-Lys-Val-Val)of Rhopilema esculentum cnidoblasts against DSS-induced UC through a combined analysis of the metagenome and serum metabolome.Specifically,the polypeptide composition of R.esculentum cnidoblasts was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF/TOF-MS).Molecular docking showed that the dominant peptide PKKVV could bind better with tumor necrosis factor-α(TNF-α)than the original ligand.Subsequent animal experiments suggested that PKKVV could modulate disorganized gut microorganisms in mice with UC;affect serum metabolites through the arachidonic acid,glycerophospholipid and linoleic acid metabolism pathways;and further alleviate UC symptoms.This study provides a reference for the comprehensive development of marine bioactive substances and nonpharmaceutical treatments for UC.

Regulation of the cellular uptake of nanopartides by the orientation of helical polypeptides

Controlling the cellular interactio n and internal izatio n of polymer-modified nan oparticles (NPs) is of central importa nee to the developme nt of promisi ng nano medici nes. Here, we describe the use of synthetic polypeptides for NP surface coati ng and regulati on of their cellular uptake behaviors by simply switching the conformation and anchoring orientation. Our results show that gold NPs (AuNPs) coated with a helical poly(Y-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)esteryl「glutamate)(L-P(EG3Glu)5o) from the C-terminus ((L-C)-AuNPs) exhibit greater zeta potential and more cellular uptake (2.0-5.5 fold higher) than those coated with the same polypeptide but anchored from the N-terminus ((L-N)-AuNPs), or from both the C- and N-terminus at a 1/1 molar ratio ((L-C/L-N)-AuNPs). A similar orientation-regulated cellular internalization pattern is observed in D-P(EG3Glu)50 but not the unstructured DL-P(EG3Glu)5o-rnodified AuNPs, suggesting an important and universal role of the helix-derived macrodipole in cellular uptake. Moreover, this orientation-governed internalization is successfully reproduced in P(EG3Glu)50-coated gold nano rods (AuNRs), and applied to the desig n of doxorubici reloaded polypeptide micelles. Simulation study offers time-resolved in sights regarding the NP-membrane in teracti ons and membrane remodeling. Thus, our study provides a delicate way of regulating the surface chemistry of NPs and the subsequent NP-cell interactions. Moreover, the results highlight the uniqueness of polypeptides in NP surface engineering, and urge a more careful consideration on the polymer orientation effect.

Selective protection of glycinebetaine on extrinsic polypeptides of PS Ⅱ particles

PHOTOSYSTEM Ⅱ（PS Ⅱ） particles capable of O2 evolution contain D1, D2 transmembrane pro-teins, and three extrinsic polypeptides which are exposed to the outer aqueous phase with ap-parent molecular masses of 33, 23, 18 kD. The three proteins can be partially or totally re-moved by treating the PS Ⅱ membranes with high concentrations of salt or other methods,

Biological effect of velvet antler polypeptides on neural stem cells from embryonic rat brain

Background Velvet antler polypeptides (VAPs), which are derived from the antler velvets, have been reported to maintain survival and promote growth and differentiation of neural cells and, especially the development of neural tissues This study was designed to explore the influence of VAPs on neural stem cells in vitro derived from embryonic rat brain Methods Neural stem cells derived from E12 14 rat brain were isolated, cultured, and expanded for 7 days until neural stem cell aggregations and neurospheres were generated The neurospheres were cultured under the condition of different concentration of VAPs followed by immunocytochemistry to detect the differentiation of neural stem cells Results VAPs could remarkablely promote differentiation of neural stem cells and most neural stem cells were induced to differentiate towards the direction of neurons under certain concentration of VAPs Conclusion Neural stem cells can be successfully induced into neurons by VAPs in vitro , which could provide a basis for regeneration of the nervous system

Synthesis,Characterization and Phase Behaviors of Polypeptides Bearing Biphenyl Mesogens and Oligo-Ethylene-Glycol Tails

A series ofpolypeptides bearing biphenyl mesogenic side-chains and oligo-ethylene-glycol （OEG） tails （PPLGn-g- BPOEGm, n = 26 and 63, m = 2, 3, and 7） has been synthesized via a 1,3-dipolar cycloaddition with quantitative grafting density. FTIR results revealed that the polypeptides adopted highly stable a-helix in the temperature range of 25-200 ℃. DSC, POM and WAXS analysis revealed that PPLGn-g-BPOEGm （m ≤ 3） samples with short OEG tail length showed two main phase transitions including crystal to liquid crystalline （smectie C, SmC） phase transition and the melting transition of crystalline E-phase, while PPLG,-g-BPOEG7 with longer OEG tail length （m = 7） exhibited the melting transitions without the formation of liquid crystalline phase.

New insights into ATR inhibition in muscle invasive bladder cancer:The role of apolipoprotein B mRNA editing catalytic subunit 3B

Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Practical guide:Glucagon-like peptide-1 and dual glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonists in diabetes mellitus

Practical guide:Glucagon-like peptide-1 and dual glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonists in diabetes mellitus common second-line choice after metformin for treating T2DM.Various considerations can make selecting and switching between different GLP-1 RAs challenging.Our study aims to provide a comprehensive guide for the usage of GLP-1 RAs and dual GIP and GLP-1 RAs for the management of T2DM.

Role of incretins and glucagon receptor agonists in metabolic dysfunction-associated steatotic liver disease:Opportunities and challenges

Metabolic dysfunction-associated steatotic liver disease(MASLD)has become the most common chronic liver disease worldwide,paralleling the rising pandemic of obesity and type 2 diabetes.Due to the growing global health burden and com-plex pathogenesis of MASLD,a multifaceted and innovative therapeutic approach is needed.Incretin receptor agonists,which were initially developed for diabetes management,have emerged as promising candidates for MASLD treatment.This review describes the pathophysiological mechanisms and action sites of three major classes of incretin/glucagon receptor agonists:glucagon-like peptide-1 receptor agonists,glucose-dependent insulinotropic polypeptide receptor agonists,and glucagon receptor agonists.Incretins and glucagon directly or indirectly impact various organs,including the liver,brain,pancreas,gastro-intestinal tract,and adipose tissue.Thus,these agents significantly improve glycemic control and weight management and mitigate MASLD pathogenesis.Importantly,this study provides a summary of clinical trials analyzing the effect-iveness and safety of incretin receptor agonists in MASLD management and provides an in-depth analysis highlighting their beneficial effects on improving liver function,hepatic steatosis,and intrahepatic inflammation.There are emerging challenges associated with the use of these medications in the real world,particularly adverse events,drug-drug interactions,and barriers to access,which are discussed in detail.Additionally,this review highlights the evolving role of incretin receptor agonists in MASLD management and suggests future research directions.